Littérature scientifique sur le sujet « Olio microbico »

Inflammatory bowel disease, inclusive of Crohn's disease and ulcerative colitis, consists of immunologically mediated disorders involving the microbiota in the gastrointestinal tract. Lavender oil is a traditional medicine used to relieve many gastrointestinal disorders. The goal of this study was to examine the therapeutic effects of the essential oil obtained from a novel lavender cultivar, Lavandula × intermedia cultivar Okanagan lavender (OLEO), in a mouse model of acute colitis caused by Citrobacter rodentium . In colitic mice, oral gavage with OLEO resulted in less severe disease, including decreased morbidity and mortality, reduced intestinal tissue damage, and decreased infiltration of neutrophils and macrophages, with reduced levels of TNF-α, IFN-γ, IL-22, macrophage inflammatory protein-2α, and inducible nitric oxide synthase expression. This was associated with increased levels of regulatory T cell populations compared with untreated colitic mice. Recently, we demonstrated that the composition of the enteric microbiota affects susceptibility to C. rodentium -induced colitis. Here, we found that oral administration of OLEO induced microbiota enriched with members of the phylum Firmicutes, including segmented filamentous bacteria, which are known to protect against the damaging effects of C. rodentium . Additionally, during infection, OLEO treatment promoted the maintenance of microbiota loads, with specific increases in Firmicutes bacteria and decreases in γ-Proteobacteria. We observed that Firmicutes bacteria were intimately associated with the apical region of the intestinal epithelial cells during infection, suggesting that their protective effect was through contact with the gut wall. Finally, we show that OLEO inhibited C. rodentium growth and adherence to Caco-2 cells, primarily through the activities of 1,8-cineole and borneol. These results indicate that while OLEO promoted Firmicutes populations, it also controlled pathogen load through antimicrobial activity. Overall, our results reveal that OLEO can protect against colitis through the microbial-immunity nexus and that a pharmacological agent, in this case OLEO, alters the normal enteric microbiota.

Herbogil (dinoterb), a reference herbicide, the mineral oil Oleo (paraffin oil used as an additive to herbicides), and Goltix (metamitron) were taken as model compounds for the study of impacts on microbial soil communities. After the treatment of soil samples, effects on metabolic sum parameters were determined by monitoring substrate-induced respiration (SIR) and dehydrogenase activity, as well as carbon and nitrogen mineralization. These conventional ecotoxicological testing procedures are used in pesticide registration. Inhibition of biomass-related activities and stimulation of nitrogen mineralization were the most significant effects caused by the application of Herbogil. Even though Goltix and Oleo were used at a higher dosage (10 times higher), the application of Goltix resulted in smaller effects and the additive Oleo was the least-active compound, with minor stimulation of test parameters at later observation times. The results served as a background for investigation of the power of “fingerprinting” methods in microbial ecology. Changes in catabolic activities induced by treatments were analyzed by using the 95 carbon sources provided by the BIOLOG system. Variations in the complex metabolic fingerprints demonstrated inhibition of many catabolic pathways after the application of Herbogil. Again, the effects of the other compounds were expressed at much lower levels and comprised stimulations as well as inhibitions. Testing for significance by a multivariate t test indicated that the sensitivity of this method was similar to the sensitivities of the conventional testing procedures. The variation of sensitive carbon sources, as determined by factor weights at different observation times, indicated the dynamics of the community shift induced by the Herbogil treatment in more detail. DNA extractions from soil resulted in a collection of molecules representing the genetic composition of total bacterial communities. Distinct and highly reproducible community patterns, or genetic fingerprints, resulting from application of the different herbicides were obtained by the sequence-specific separation of partial 16S rDNA amplification products in temperature gradient gel electrophoresis. Significant pattern variations were quantified. For detailed analysis, application-responsive bands from the Herbogil and Oleo treatments were sequenced and their tentative phylogenetic positions were identified. Data interpretation and the potentials and biases of the additional observation windows on microbial communities are discussed.

Nutrient and organic matter concentration, microbial biomass and activities were studied at the surface microlayers (SML) and subsurface waters (SSW) in two small forest lakes of different water colour. The SML in polyhumic lake is more enriched with dissolved inorganic nitrogen (0.141 mg l–1) than that of oligohumic lake (0.124 mg l–1), the former also contains higher levels of total nitrogen (2.66 mg l–1). Higher activities of lipase (Vmax 2290 nmol l–1 h–1 in oligo- and 6098 in polyhumic) and glucosidase (Vmax 41 nmol l–1 h–1 in oligo- and 49 in polyhumic) were in the SMLs in both lakes. Phosphatase activity was higher in the oligohumic SML than in SSW (Vmax 632 vs. 339 nmol l–1 h–1) while in polyhumic lake was higher in SSW (Vmax 2258 nmol l–1 h–1 vs. 1908 nmol l–1 h–1). Aminopeptidase activity in the SSW in both lakes was higher than in SMLs (Vmax 2117 in oligo- and 1213 nmol l–1 h–1 in polyhumic). It seems that solar radiation does inhibit neuston microbial community as a whole because secondary production and the share of active bacteria in total bacteria number were higher in SSW. However, in the oligohumic lake the abundance of bacteria in the SML was always higher than in the SSW (4.07 vs. 2.69 × 106 cells ml–1) while in the polyhumic lake was roughly equal (4.48 vs. 4.33 × 106 cells ml–1) in both layers. Results may also suggest that surface communities are not supplemented by immigration from bulk communities. The SML of humic lakes may act as important sinks for allochthonous nutrient resources and may then generate considerable energy pools for microbial food webs.

Microbial degradation of natural rubber and synthetic poly(cis-1,4-isoprene) is expected to become an alternative treatment system for waste from poly(cis-1,4-isoprene) products including scrap tires. Nocardia farcinica NBRC 15,532, a gram-positive rubber-degrading bacterium, can utilize poly(cis-1,4-isoprene) as the sole source of carbon and energy to produce oligo-isoprene metabolites containing aldehyde and keto end groups. A homology-based search of the genome revealed a gene encoding a latex-clearing protein (Lcp). Gene disruption analysis indicated that this gene is essential for the utilization of poly(cis-1,4-isoprene) in this strain. Further analysis of the genome sequence identified aldehyde dehydrogenase (ALDH) genes as potential candidates for oxidative degradation of oligo-isoprene aldehydes. Based on the enzymatic activity of the ALDH candidates, NF2_RS14000 and NF2_RS14385 may be involved in the degradation of oligo-isoprene aldehydes. Analysis of the reaction products revealed that these ALDHs oxidized tri- to penta-isoprene aldehydes, which were generated by the reaction of Lcp. Based on the inability of ALDH gene deletion mutants, we concluded that NF2_RS14000 is mainly involved in the utilization of poly(cis-1,4-isoprene) and the oxidative degradation of oligo-isoprene aldehydes in Nocardia farcinica NBRC 15,532.

Many environmental and biomedical biomonitoring and detection studies aim to explore the presence of specific organisms or gene functionalities in microbiome samples. In such cases, when the study hypotheses can be answered with the exploration of a small number of genes, a targeted PCR-approach is appropriate. However, due to the complexity of environmental microbial communities, the design of specific primers is challenging and can lead to non-specific results. We designed PhyloPrimer, the first user-friendly platform to semi-automate the design of taxon-specific oligos (i.e., PCR primers) for a gene of interest. The main strength of PhyloPrimer is the ability to retrieve and align GenBank gene sequences matching the user’s input, and to explore their relationships through an online dynamic tree. PhyloPrimer then designs oligos specific to the gene sequences selected from the tree and uses the tree non-selected sequences to look for and maximize oligo differences between targeted and non-targeted sequences, therefore increasing oligo taxon-specificity (positive/negative consensus approach). Designed oligos are then checked for the presence of secondary structure with the nearest-neighbor (NN) calculation and the presence of off-target matches with in silico PCR tests, also processing oligos with degenerate bases. Whilst the main function of PhyloPrimer is the design of taxon-specific oligos (down to the species level), the software can also be used for designing oligos to target a gene without any taxonomic specificity, for designing oligos from preselected sequences and for checking predesigned oligos. We validated the pipeline on four commercially available microbial mock communities using PhyloPrimer to design genus- and species-specific primers for the detection of Streptococcus species in the mock communities. The software performed well on these mock microbial communities and can be found at https://www.cerealsdb.uk.net/cerealgenomics/phyloprimer.

“Boswellia serrata” is a herb used for ages in the Indian system of medicine and other systems of medicines across the world. It comes under the family “Burseraceae”. The Boswellia serrata is called by various local names in different regions, out of which “Kundur” “salai” “guggul” “Shallaki” are very prominently used. “Shallaki” is a widely used active ingredient in the “Unani” “Siddha” and “Ayurvedic” medicines oriented for the treatment of inflammation and pain. It is also used as a component to add fragrance to the products. Boswellia serrata plant contains oleo-gum resins and the ASU drug manufacturer mostly use this resin to prepare Boswellia-oriented drugs and this practice of using resin as the key component of drugs is very old and effective. In ancient ayurvedic texts, particular methods describe the extraction, purification, and usage of this oleo gum resin. Now in the modern era, when a lot of research has been done on this plant and its parts, this plant has come out to be even more important as it is found to be showing specific characteristics beneficial for treating illnesses such as dysentery, urinary tract disorders, hemorrhoids, ulcers, and dyspepsia. The present review provides an overview of some pharmacological activities and the importance of “Salaiguggal” or “ Boswellia serrata “. “Salai guggal” is rich in essential oils, and therefore, it becomes important for the perfume industries as its essential oils are soothing to smell and show therapeutic effects. The essential oils of Boswellia serrata are a mixture of different boswellic acids such as “mono-terpenes”, “di-tepenes” and “sesqui-terpenes”. Whereas the oleo gum resin of “ Boswellia serrata “ comprises monosaccharides such as pentose and hexose sugars. The oleo gum resin of this plant is highly recommended by almost every medical practitioner who deals in herbal medicines as it has shown a huge range of effective characteristics in asthma, cancer, microbial/fungal infections, inflammation, arthritis, diarrhea, and also as an analgesic. A proper systematic literature review was done using different databases that were available online like Google Scholar, PubMed, and ScienceDirect.

Nel settore degli pneumatici, comunemente vengono utilizzati oli minerali, derivanti dal petrolio, come plastificanti. La tossicità delle loro componenti, insieme alla diminuzione della loro disponibilità e accessibilità, sta causando problemi ambientali e politici. Alcuni oli vegetali, composti in generale da una miscela trigliceridi, sono stati testati con successo come alternativa. Sfortunatamente, anche questi oli pongono problemi ambientali, economici e sociali, come materiali di partenza. I lieviti oleaginosi, oltre ad essere capaci di accumulare oli con una composizione simile a quella degli oli vegetali, posseggono diverse caratteristiche: a)accumulo di oli fino al 70% del loro peso secco cellulare, in opportune condizioni di crescita; b) coltivazione su larga scala; c) utilizzo di biomasse residuali ,biomasse di seconda generazione, come substrato per la loro crescita e accumulo di oli; d) possibile utilizzo di tecniche di ingegnerizzazione per modulazione composizione finale degli oli, rendendoli una alternativa promettente per la produzione di oli plastificanti.Nella prima parte di questo lavoro, sono stati prodotti due diversi oli microbici, MICRO-OIL1 (MO1) e 2 (MO2), a partire da due lieviti oleaginosi R. toruloides e L. starkeyi, attraverso fermentazione utilizzando come substrato per la loro crescita ed accumulo un prodotto di scarto derivante dall’industria del biodiesel (glicerolo grezzo). In seguito, sono stati testati all’interno di mescole pneumatiche, a base di butadiene e gomma naturale, per verificare il loro effetto sulle proprietà. Tali oli, aventi diversa composizione in termini di acidi grassi, hanno permesso in generale di ottenere delle proprietà statiche e dinamiche molto simili al riferimento minerale, MES, e migliori rispetto agli oli vegetali utilizzati come riferimento, conferendo inoltre bassa resistenza al rotolamento, attribuibile alla riduzione dei valori di tanδ alla temperatura di 70°. In questa parte, è stato inoltre dimostrato come la presenza degli antiossidanti naturali, carotenoidi, all’interno del MO1, sia in grado di far mantenere le proprietà statiche alle mescole pneumatiche anche dopo invecchiamento termico, suggeriscono un’attività protettiva del MO1.Nella seconda parte del lavoro, studi di compatibilità basati su analisi termica con Dynamic Scanning Calorimetry (DSC), effettuati miscelando gli oli (MO1, MO2, AP-88 e MES) e due diversi elastomeri a base di stirene-butadiene, hanno rivelato l’affinità tra il MO2 e l’elastomero SLR3402, confermata anche in mescola pneumatica, da proprietà statiche e dinamiche simili al riferimento, MES, e migliori rispetto al riferimento vegetale, AP-88.Nell’ultima parte di questo lavoro, è stato adottato un approccio di ingegneria metabolica, con il fine di dimostrare il potenziale di tali lieviti come produttori di oli plastificanti. Con questo intento sono state condotte sul lievito oleaginoso L. starkeyi ingegnerizzazioni geniche mirate a ridirezionare il flusso degli acidi grassi prodotti. A tale scopo, sono stati overespressi i geni OLE1 e FAD2, codificanti rispettivamente per la Δ9 e Δ12 desaturasi, coinvolti principalmente nella produzione di acidi grassi monoinsaturi (MUFAs) e poliinsaturi (PUFAs). I risultati ottenuti hanno mostrato come la combinata overespressione conduca verso una ridistribuzione degli acidi grassi in favore dei MUFAs all’interno dell’olio microbico, MICRO-OIL4 (MO4), in seguito, anche testato in mescole pneumatiche a base di SLR3402. Sono state ottenute proprietà statiche e dinamiche simili al riferimento MES, con carico a rottura migliore rispetto ad AP-88, e riduzione dei valori di tanδ a 70° indicando una bassa resistenza al rotolamento. In conclusione, le buone performance osservate da incorporazione di oli microbici in mescole pneumatiche, suggeriscono come essi possano utilizzati come potenziali plastificanti per rimpiazzare i tradizionali oli del settore pneumatico.
In the field of tyre, it is a common practice to use mineral oils, petroleum-derived, as plasticizers. The toxicity of their components, together with their diminishing availability and/or accessibility, is causing environmental and political concerns. Some vegetable oils, consisting of Triacylglycerols (TAG) mixture, have been successfully tested as alternative. Unfortunately, also these oils pose environmental, economic and social issues, as the starting material. The oleaginous yeasts, together with their capability to accumulate microbial oils, with a composition similar to vegetable oils, have different characteristics as: a) are able to accumulate oil up to 70% of their dry cell weight, under proper conditions; b) large-scale cultivation; b)use of residual biomasses, as cultivation and oil accumulation substrate; d)genetic tools available in order to modify the final oil composition, make them promising alternative for plasticizing oils production. In the first part of this work, it has been described the production of two different microbial oil, MICRO-OIL1 (MO1) e 2 (MO2), starting from two oleaginous yeasts R. toruloides and L. starkeyi, by fermentation using as substrate, for their growth and oil accumulation, a waste product of biodiesel production (crude glycerol). Then, the resulting oils have been tested in rubber compounds, butadiene-natural rubber based, to verify their effect on their properties. The oils, having different fatty acids compositions, allow to obtain static and dynamic properties similar to the reference mineral oil, MES, and better than the vegetable oils using as reference, giving, in addition lower rolling resistance thanks to the lower value of tanδ at 70°. In this part, it was also demonstrated as the presence in the MO1 of natural antioxidant, carotenoids, allows to the rubber compounds to maintain the static properties also after thermal aging, allowing to hypnotize a protective action of MO1. In the second part of this work, compatibility studies based on thermal analysis by Dynamic Scanning Calorimetry (DSC), carried out mixing the oils (MO1, MO2, AP-88 e MES) and two different elastomers, styrene-butadiene based, revealed the affinity between MO2 and the elastomer SLR3402, also confirmed by the static and dynamic properties of the rubber compound, that resulting similar to the reference oil, MES, and better than vegetable oil, AP-88. In the last part of this work, has been adopted a metabolic engineering approach to demonstrate the potential of these yeasts as plasticizer producers. To this purpose, the oleaginous yeast L. starkeyi has been engineered in order to redirect the fatty acids flux production. Then, two genes OLE1 and FAD2, encoding for two enzymes Δ9 e Δ12 desaturase, involved in the production of mono- (MUFAs) and polyunsaturated (PUFAs) fatty acids. The results showed as the combined overexpression led to a redistribution of fatty acids in favor of MUFAs inside the microbial oil, the MICRO-OIL4 (MO4), also tested in rubber compound SLR3402-based. The compound showed static and dynamic properties similar to MES, a better tensile strength value compared to AP-88, as well as a reduction of tanδ at 70°, that led to lower rolling resistance. The good performance obtained by microbial oil incorporation in rubber compounds, suggesting as they can be used as potential plasticizing to replace the traditional oils used in tyre field.

O tradicional biodiesel de 1ª geração (produzido a partir de óleo de origem vegetal, como soja ou canola) possui muitas desvantagens e limitações como sazonalidade, uso de grandes áreas de cultivo, competição com alimentos, dentre outras. Uma alternativa são os óleos produzidos por microrganismos. Com o objetivo de otimizar a produção do óleo microbiano, o presente trabalho avaliou a produção de biomassa, lipídios e composição de ácidos graxos da levedura Yarrowia lipolytica QU21 quando cultivada em diferentes fontes de carbono (glicose e glicerol), nitrogênio (sulfato de amônio, triptona, ureia, nitrato de amônio e extrato de levedura), assim como diferentes condições de cultivo (agitação, aeração e razão carbono/nitrogênio). Dois resíduos industriais, glicerina bruta e resíduo de indústria cervejeira (FYE) também foram testados como substitutos da fonte de carbono e nitrogênio, respectivamente. Este trabalho também apresenta uma técnica de triagem de leveduras oleaginosas, de forma a quantificar os lipídios utilizando solventes menos agressivos, tanto para o manipulador quanto para o meio ambiente. A composição de ácidos graxos do óleo produzido pela Y. lipolytica QU21 quando cultivada em glicerina bruta e sulfato de amônio apresentou potencial utilização como matéria prima para o biodiesel. O uso combinado dos dois resíduos industriais pela Y. lipolytica QU21 resultou na produção de óleo com elevado teor de ácidos graxos poliinsaturados. Além de tornar o óleo microbiano da levedura Y. lipolytica QU21 uma matéria prima competitiva para a produção de biodiesel, a utilização da glicerina bruta poderia atenuar problemas ambientais, como a disposição inadequada no meio ambiente.
The traditional 1st generation biodiesel (produced from plant oils, such as soybeans and canola) has many drawbacks and limitations as season and climate-dependent cultivation, agricultural land competition for food, among others. Possible alternative oil sources is microbial oil produced by oleaginous microorganisms. With the purpose of optimizing the production of microbial oil, this study evaluated the production of biomass, lipid and fatty acid composition of the yeast Yarrowia lipolytica QU21 when grown on different carbon source (glucose and glycerol), nitrogen source (ammonium sulfate, tryptone, urea, ammonium nitrate and yeast extract) as well as different culture conditions (agitation, aeration and carbon/nitrogen ratio). Two industrial waste were also evaluated, crude glycerol and brewery waste (FYE) as surrogate carbon and nitrogen sources, respectively. This work also presents a technique for sorting oleaginous yeast in order to quantify the lipids using less aggressive solvent for both the handler and to the environment. The fatty acid composition of the oil produced by the Y. lipolytica QU21 growing on crude glycerol and ammonium sulfate showed potential use as a feedstock for biodiesel. The combined wastes resulted on microbial oil produced by Y. lipolytica QU21 with high polyunsaturated fatty acid content. Besides making the microbial oil a competitive feedstock for biodiesel production, the use of crude glycerol could mitigate environmental issues such as improper waste disposal.

Gli oli microbici stanno ricevendo sempre più attenzioni come possibile alternativa agli oli vegetali, nel processo di sostituzione dei combustibili fossili. Tuttavia, diversi aspetti necessitano di essere ottimizzati al fine di ottenere oli economicamente competitivi e con caratteristiche chimico-fisiche desiderate. In questa ricerca, sono stati utilizzati due differenti approcci per poter realizzare l’obiettivo preposto. Il primo, si è basato sull’ingegnerizzazione genetica del lievito C. oleaginous, al fine di incrementare la produttività di lipidi e modificare la composizione dei trigliceridi (TAG) sintetizzati. Un protocollo basato su una trasformazione genetica mediata da Agrobacterium è stato utilizzato per sovraesprimere la diacilglicerol trasnferasi (DGA1), l’enzima responsabile dell’ultimo step della sintesi dei TAG, e la Δ9-desaturasi, l’enzima che catalizza la conversione dell’acido stearico (C18:0) in acido oleico (C18:1). La selezione di colonie positive e l’analisi dei mutanti ottenuti ha confermato la buona riuscita della trasformazione. Il secondo approccio ha mirato a studiare l’influenza sulla crescita e sul profilo di lipidi accumulati da C. oleaginous da parte di diversi acidi grassi volatili (VFAs), una materia prima ottenibile da trattamenti di scarti industriali. A questo proposito, sono state utilizzate fermentazioni fed-batch su scala da 1-L basate su glucosio e miscele sintetiche di acido acetico e di VFAs come fonte di carbonio. L’utilizzo simultaneo di acido acetico e acidi secondari ha mostrato come sia possibile stimolare il metabolismo microbico al fine di incrementare l'accumulo di oli e ottenere una composizione chimica lipidica desiderata.

Encara que els gens de resistència a antibiòtics són una eina important per a la selecció i manteniment de plàsmids recombinants, les autoritats reguladores consideren el seu ús inacceptable en diverses àrees dins la indústria biotecnològica, sobretot quan el producte serà utilitzat amb finalitat terapèutica. Un sistema d’expressió basat en el vector pQE (Quiagen) amb un marcador de selecció plasmídic alternatiu (basat en l’auxotròfia de glicina) va ser desenvolupat prèviament (Vidal et al., 2008). El sistema d’expressió pQE es basa en el promotor T5, induïble per IPTG. La no-­‐ estabilitat del promotor en absència d’inductor pot provocar una inestabilitat estructural del vector, que portarà a nivells baixos d’expressió deguts, per exemple, a fenòmens de recombinació a la regió del promotor T5. El repressor lac, codificat pel gen lacI, s’uneix molt fortament al promotor, interferint en la transcripció del gen d’interès. Així doncs, els nivells transcripcionals del gen lacI tenen un paper crític en els sistemes d’expressió basats en el promotor T5, influenciant els nivells basals de transcripció i la concentració d’inductor. Encara que la sobreexpressió de la proteïna d’interès és un factor important en quant a càrrega metabòlica, també cal tenir en compte la contribució deguda a l’expressió d’altres gens plasmídics. En aquest sentit, per tal de millorar la robustesa del sistema i minimitzar la càrrega metabòlica relacionada amb els gens codificats als plàsmids, s’han eliminat tots els gens de resistència a antibiòtics i s’han afinat els nivells d’expressió del marcador auxotròfic (glyA) i del repressor lac (lacI). En aquest treball s’han construït uns cassettes d’expressió on els gens lacI i glyA estan sota el control d’un seguit de promotors constituents sintètics, per tal d’obtenir suficient inhibidor lacI per evitar la “no-­‐estabilitat del promotor” i assegurar el nivell mínim de transcripció de glyA necessaris tant per al manteniment del plàsmid com per al correcte creixement del cultiu en medi definit. A més, el gen de resistència a antibiòtic en el vector d’expressió va ser reemplaçat per la construcció glyA-­‐lacI, obtenint un sistema totalment lliure de gens per a la resistència a antibiòtic. Finalment, s’ha estudiat la capacitat per a la sobreexpressió de FucA recombinant a diferents escales (flascons agitats i bioreactor) i en diversos modes d’operació (discontinu i discontinu alimentat) per tal d’obtenir alts nivells d’expressió. Per últim, per tal d’analitzar la població bacteriana a nivell de cèl·∙lules individuals, els canvis morfològics (FSC i SSC) i les característiques físiques i bioquímiques de les diferents cèl·∙lules dins la població bacteriana, s’han realitzat anàlisis de citometria de flux.
While antibiotic resistance marker genes are a powerful system for selection and maintenance of recombinant plasmids in hosts such as E. coli, its use has been considered unacceptable in many areas of biotechnology by regulatory authorities, particularly when the recombinant product will be used in the therapeutic field. Previously, we have developed an expression system based on the pQE vector series (Qiagen) with an alternative plasmid selection marker based on glycine auxotrophy complementation (Vidal et al., 2008). The pQE expression system is based on the IPTG-­‐induced T5 promoter. Promoter leakiness in inducer absence may lead to structural instability of the expression vector, resulting in reduced expression levels, e.g. due to recombination events in the T5 promoter region. The lac repressor, encoded by the lacI gene, binds very tightly to the promoter, interfering with the transcription of the gene of interest. Therefore, lacI transcriptional level plays a key role in T5 promoter-­‐based expression systems, influencing the basal transcriptional levels and the concentration of inducer. Furthermore, although the overexpression of the plasmid-­‐encoded protein of interest is a major factor in the metabolic burden, expression of other plasmid-­‐genes may also contribute. Thus, in order to improve the system robustness, all the antibiotic resistance genes have been removed and the expression levels of the auxotrophic marker (glyA) and the lac repressor (lacI) genes have been fine-­‐tuned, in order to minimize the metabolic burden related to plasmid-­‐encoded genes. In this study, an expression cassette has been constructed where the lacI and glyA genes have been placed under the control of selected synthetic constitutive promoters in order to obtain the sufficient lacI inhibitor ensuring lack of “promoter leakiness” and the minimal glyA transcriptional levels needed for plasmid maintenance and optimal cell growth in defined media. Moreover, in the expression vector the antibiotic resistance gene was replaced by the lacI-­‐glyA cassette, thus yielding a completely antibiotic resistance gene free system. Finally, in order to obtain high expression levels of the protein of interest, the capacity for recombinant FucA overexpression has been investigated at different scales (shake flasks and bioreactors) and operation modes, batch and fed-­‐batch. Lastly, flow cytometry analyses were performed in order to analyze the bacterial populations at the single-­‐cell level: changes in morphology (FSC and SSC) and physical and biochemical characteristics of individual cells within a bacterial population.

La fermentació alcohòlica la realitza una comunitat microbiana complexa, on els llevats vínics juguen un paper important. En els darrers anys, hi ha hagut un creixent interès per millorar complexitat del vi mitjançant fermentacions controlades quer utilitzaven no només S. cerevisiae sinó també soques seleccionades de llevats no-Saccharomyces. La investigació sobre la viabilitat dels llevats i les interaccions tenen un paper fonamental per entendre la diversitat de llevats en la fermentació mixta. En aquesta tesi, les tècniques independents de cultiu que es van aplicar per a l'anàlisi directa de mostres de vins inclouen la seqüenciació massiva, fluorescència per hibridació in situ (FISH) en combinació amb microscòpia i citometria de flux, RT-qPCR i EMA-DGGE. Aquestes tècniques independents de cultiu permeten una ràpida identificació i / o quantificació dels diferents llevats del vi. Aquestes tècniques han estat utilitzades en fermentacions espontànies a la regió del Priorat, i H. uvarum i Starm. bacillaris van ser les dues principals espècies de llevats no-Saccharomyces aïllades. H. uvarum o Starm. bacillaris perden gradualment la seva cultivabilitat quan els mostos es van inocular amb S. cerevisiae, però es podien quantificar cèl•lules de llevats en estat viable però no cultivable. La pèrdua de cultivabilitat dels no Saccharomyces va ser induïda principalment per alguns metabòlits secretats per S. cerevisiae, però els canvis en altres metabòlits principals també van influir. Aquesta interacció de llevat no Saccharomyces amb S. cerevisiae era específica d’espècie i soca.
La fermentación alcohólica es llevada a cabo por una comunidad microbiana compleja, donde las levaduras del vino juegan un papel importante. En los últimos años, ha habido un creciente interés para mejorar la complejidad del vino en fermentaciones controladas utilizando no sólo S. cerevisiae sino también algunas cepas seleccionadas de levaduras no-Saccharomyces. La investigación sobre la viabilidad de las levaduras y las interacciones tienen un papel fundamental para entender la diversidad de levaduras en fermentaciones mixtas. En esta tesis, se aplicaron técnicas independientes de cultivo para el análisis de muestras de vinos directa incluyendo la secuenciación masiva, fluorescencia por hibridación in situ (FISH) en combinación con microscopía y citometría de flujo, RT-qPCR y EMA-DGGE. Estas técnicas independientes de cultivo permiten una rápida identificación y / o cuantificación de las distintas levaduras del vino. Estas técnicas han sido utilizadas para el análisis de fermentaciones espontáneas en la región del Priorat, siendo las especies H. uvarum y Starm. bacillaris las dos principales especies de levaduras no-Saccharomyces. H. uvarum o Starm. bacillaris pierden gradualmente su cultivabilidad cuando los mostos se inocularon con S. cerevisiae, pero las levaduras se pudieron cuantificar en estado viable pero no cultivable. La pérdida de cultivabilidad de las especies no Saccharomyces fue inducida principalmente por algunos metabolitos secretados por S. cerevisiae, pero los cambios en otros metabolitos principales también influyen. Esta interacción entre levaduras no Saccharomyces con S. cerevisiae es especie y cepa dependiente.
Alcoholic fermentation is driven by complex microbial community, where wine yeasts play an important role. In recent years, there has been growing interest to enhance wine complexity by controlled fermentations using not only S. cerevisiae but also together with some selected non-Saccharomyces yeast strains. Research on yeast viability and interaction has a fundamental role to understand the diversity of yeast in mixed fermentations. In this thesis, culture-independent techniques were developed and applied for direct wine sample analysis including massive sequencing, fluorescence in situ hybridization (FISH) combined with microscopy and flow cytometry, RT-qPCR and EMA-DGGE. These culture-independent techniques enable fast identification and/or quantification of different wine yeasts. These techniques have been used during spontaneous fermentation in Priorat region, and H. uvarum and Starm. bacillaris where the two main non-Saccharomyces yeast species detected. H. uvarum or Starm. bacillaris gradually lost their culturability when musts were inoculated with S. cerevisiae, but quantifiable yeast cells existed in viable but non-culturable state. The culturability loss of non-Saccharomyces was mainly induced by some metabolites secreted from S. cerevisiae, but changes in other main metabolites also had some effect. This interaction of non-Saccharomyces yeast with S. cerevisiae showed the specificity of species and strains.

El efecto de las tasas de compresión y descompresión a altas presiones hidrostáticas (HHP) en la inactivación de bacterias ha sido poco estudiado. La literatura disponible presenta resultados contradictorios. Este estudio se diseñó para analizar el efecto de diferentes tasas de compresión y descompresión durante los tratamientos de HHP sobre matrices seleccionadas (tampón Tris, leche desnatada y zumo de naranja) inoculados con cepas bacterias seleccionadas en estado vegetativo (Escherichia coli y Staphylococcus aureus) y formadoras de esporas (Bacillus subtilis). Los experimentos HHP se llevaron a cabo utilizando diferentes máquinas en diferentes centros de investigación y las muestras se analizaron por procedimientos microbiológicos y biotecnológicos. Los resultados revelaron que el aumento de la inactivación de las bacterias vegetativas se puede lograr mediante el uso de la compresión rápida durante el procesamiento HHP. La compresión lenta se supone que induce una respuesta de estrés en las células microbianas que conduce a un menor efecto de inactivación del proceso. Por otro lado, las esporas bacterianas son más sensibles a la ralentización de las tasas de compresión durante el procesamiento HHP. Bajo nuestras condiciones experimentales, la descompresión lenta dio como resultado una mayor inactivación de las bacterias formadoras de esporas y de las células vegetativas en comparación con la descompresión rápida. Una descompresión lenta se relaciona con un tiempo de procesamiento prolongado, causa las células ya han recibido un daño subletal durante la fase de compresión y de mantenimiento de presión resultan más sensibles al tratamiento prolongado. Estudios adicionales realizados para investigar los daños subletales, revelaron que la compresión rápida y descompresión lenta causaron un mayor número de células con daño subletal en todos los microorganismos estudiados. Una parte de esta población con daño subletal es capaz de recuperar sus lesiones en el entorno de baja acidez (como la leche y el tampón Tris) y puede comprometer la seguridad alimentaria. Si bien en el ambiente de elevada acidez (como el jugo de naranja), estas bacterias subletal no pueden recuperarse y terminan muriendo durante los15 días de almacenamiento.
The effect of compression and decompression rates of high hydrostatic pressure (HHP) for inactivation of bacteria has been scarcely studied. The available literature presented contradictory results. This study was designed to analyze the effect of different rates of compression and decompression during HHP treatments of selected matrices (Tris buffer, skimmed milk and orange juice) inoculated with selected strains of vegetative (Escherichia coli & Staphylococcus aureus) and spore forming (Bacillus subtilis) bacteria. The HHP experiments were conducted using different HHP machines in different research centers and samples were analyzed by microbiological and biotechnological procedures. Results revealed that higher inactivation of vegetative bacteria can be achieved by using fast compression rates during HHP processing. While slow compression is supposed to induce a stress response in microbial cells that leads to lower inactivation effect of the process. On the other hand, bacterial spores are more sensitive to slow compression rates during HHP processing. Under our experimental conditions slow decompression resulted in higher inactivation of vegetative and spore forming bacteria as compared to fast decompression. Slow decompression is attributed with extended processing time. When cells are sublethally injured by compression treatments and pressure holding time, they become more sensitive to upcoming processing stages. Further studies to investigate the sublethal injuries, revealed that fast compression and slow decompression resulted in highest number of sublethally injured cells in all microorganisms. A part of this sublethally injured population is able recover their injuries in low acid environment (like milk and Tris buffer) and may challenge the food safety. While in high acid environment (like orange juice), these sublethally injured bacterial are unable to recover and become dead during 15 days storage